Compositions and methods for modulation of LMNA expression

ABSTRACT

Disclosed herein are compounds, compositions and methods for modulating the expression of LMNA in a cell, tissue or animal. Also provided are methods of target validation. Also provided are uses of disclosed compounds and compositions in the manufacture of a medicament for treatment of diseases and disorders. Further provided are methods of identifying cis splicing regulatory elements of a selected mRNA using the disclosed compounds.

RELATED APPLICATIONS

This application is the U.S. National Phase under 35 U.S.C. §371 ofInternational Application PCT/US2006/041018, filed Oct. 17, 2006, whichapplication claims the benefit of priority to U.S. ProvisionalApplication Ser. No. 60/728,709, filed Oct. 20, 2005 and U.S.Provisional Application Ser. No. 60/754,517, filed Dec. 27, 2005, all ofwhich are herein incorporated by reference in their entirety.

BACKGROUND

The LMNA gene encodes two alternatively spliced products, lamin A andlamin C (Lin and Worman, 1993, J. Biol. Chem. 268:16321-16326). TheN-terminal 566 amino acids of lamin A and lamin C are identical withlamin C containing 6 unique amino acids at the C-terminus to yield aprotein of 572 amino acids. Lamin A, which is 646 amino acids in length,is generated from a precursor protein, prelamin A, by a series ofposttranslational processing steps (Young et al., 2005, J Lipid Res. Oct5 electronic publication). The first step in prelamin A processing isfarnesylation of a carboxyl-terminal cysteine residue, which is part ofa CAAX motif at the terminus of the protein. Next, the terminal threeamino acids (AAX) are cleaved from the protein, after which thefarnesylcysteine is methylated. Finally, the C-terminal 15 amino acidsare enzymatically removed and degraded to form mature lamin A.

Lamin A and lamin C are key structural components of the nuclear lamina,an intermediate filament meshwork underneath the inner nuclear membrane.The lamin proteins comprise N-terminal globular head domains, centralhelical rod domains and C-terminal globular tail domains. Lamins A and Chomodimerize to form parallel coiled-coil dimers, which associatehead-to-tail to form strings, and ultimately form the higher-orderfilamentous meshwork that provides structural support for the nucleus(Muchir and Worman, 2004, Physiology (Bethesda) 19:309-314; Mutchisonand Worman, 2004, Nat. Cell Biol. 6:1062-1067; Mounkes et al. 2001,Trends Cardiovasc. Med. 11:280-285).

Hutchinson-Gilford progeria syndrome (HGPS) is a childhood prematureaging disease resulting from the production of a mutant form offarnesyl-prelamin A, which cannot be processed to mature lamin A. Theaccumulation of farnesyl-prelamin A is toxic, inducing misshapen nucleiat the cellular level and a wide range of disease symptoms at theorganismal level (e.g., osteoporosis, alopecia, micrognathia and dentalabnormalities). HGPS is most commonly caused by a spontaneous mutationin exon 11 of LMNA, which activates a cryptic splice site fournucleotides upstream of the mutation (a cytosine to thyrmidinesubstitution at codon 608) (Eriksson et al. 2003, Nature 423:293-298).The pre-mRNA derived from the mutated allele is spliced using theaberrant donor splice site and the correct exon 12 acceptor site,yielding a truncated LMNA mRNA lacking the terminal 150 nucleotides ofexon 11. As a result of aberrant splicing, a mutant protein lacking 50amino acids from the globular tail is produced.

Antisense compounds targeting a selected mRNA or pre-mRNA molecule haveproven effective at either reducing total levels of target mRNA throughtarget degradation, or altering the ratio of specific target spliceproducts through occupancy-based mechanisms. Given the role of LMNA indiseases such as HGPS, methods of reducing expression of LMNA mRNA ormethods of modulating splicing of LMNA pre-mRNA to eliminate expressionof mutant lamin A protein are needed.

A method of controlling the behavior of a cell through modulation of theprocessing of an mRNA target by contacting the cell with an antisensecompound acting via a non-cleavage event is disclosed in U.S. Pat. No.6,210,892 and U.S. Pre-Grant Publication 2002-0049173.

Kole et al. (WO 94/26887 and U.S. Pat. Nos. 5,627,274; 5,916,808;5,976,879; and 5,665,593) disclose methods of combating aberrantsplicing using modified antisense oligonucleotides which do not activateRNase H.

Scaffidi and Misteli (2005, Nat. Med. 11(4):440-445) disclose amorpholino oligonucleotide used to correct aberrant splicing of LMNA inHGPS fibroblasts.

U.S. Pre-Grant Publication 2005-0059071 discloses mutations in the LMNAgene that cause HGPS and methods of influencing expression of LMNA. Suchmethods include the use of oligonucleotides and other compounds.

Huang et al (2005, Human Genet., Oct 6, electronic publication) discussshort hairpin RNA (shRNA) constructs designed to target mutant LMNApre-mRNA or mature LMNA mRNA to decrease expression of mutant lamin Aprotein.

Harborth et al. (2003, Antisense Nucleic Acid Drug Dev. 13(2):83-105)discuss LMNA gene silencing using siRNA compounds.

Antisense technology is an effective means for reducing the expressionof one or more specific gene products and is uniquely useful in a numberof therapeutic, diagnostic, and research applications. Provided hereinare antisense compounds for use in modulation of LMNA expression, eitherthrough RNase H-dependent cleavage, or by modulation of LMNA splicing.Also provided herein is a method for identifying cis splicing regulatoryelements of a selected pre-mRNA, such as LMNA pre-mRNA.

SUMMARY

Provided herein are antisense compounds targeted to LMNA whichspecifically hybridize with and inhibit expression of LMNA. In oneembodiment, the antisense compounds comprise at least one2′-O-(2-methoxyethyl) nucleotide. Preferred antisense compounds areantisense oligonucleotides. In one embodiment, the antisenseoligonucleotides provided herein comprise at least one modifiedinternucleoside linkage. Also provided are antisense oligonucleotidescomprising modified internucleoside linkages at each position. In oneaspect, the modified internucleoside linkage is phosphorothioate. In oneembodiment, the antisense oligonucleotides comprise at least onemodified nucleobase. In one aspect, the modified nucleobase is5-methylcytosine.

Further provided are chimeric antisense oligonucleotides targeted toLMNA. In one embodiment, the chimeric antisense oligonucleotidescomprise a first region with one or more deoxynucleotides and second andthird regions flanking said first region, each with at least one2′-O-(2-methoxyethyl) nucleotide. In one aspect, the first regioncontains 10 nucleotides and each of the second and third regions contain5 nucleotides. The chimeric antisense oligonucleotides provided hereincan further comprise modified internucleoside linkages and/or modifiednucleobases.

Also provided herein are pharmaceutical compositions comprising theantisense compounds and a pharmaceutically acceptable penetrationenhancer, carrier or diluent.

Methods of inhibiting expression of LMNA in cells or tissues bycontacting the cells or tissues with the antisense compounds describedherein are also provided.

Also provided herein are antisense oligonucleotides targeted to LMNApre-mRNA which specifically hybridize with and modulate processing ofLMNA. In one embodiment, the antisense oligonucleotides comprise amodified sugar residue at each nucleotide. In one aspect, the modifiedsugar moiety is 2′-O-(2-methoxyethyl). In another embodiment, theantisense oligonucleotides that modulate processing comprise a modifiedinternucleoside linkage at each position. In another embodiment, eachcytosine of the antisense oligonucleotide is replaced with5-methylcytosine. In one aspect, the modulation of processing ismodulation of splicing. In one embodiment, the antisenseoligonucleotides are targeted to intron:exon, exon:intron or exon:exonjunctions. In another embodiment, the antisense oligonucleotides targeta splice site. Splice sites include, but are not limited to, spliceacceptor sites, splice donor sites, constitutive splice sites, crypticsplice sites and aberrant splice sites. In another embodiment, theantisense oligonucleotides target cis splicing regulatory elements.Regulatory elements include, but are not limited to, intronic splicingenhancers, intronic splicing silencers, exonic splicing enhancers andexonic splicing silencers. In another embodiment, the antisenseoligonucleotides target exon 11 of LMNA. In one aspect, the antisenseoligonucleotides that modulate splicing target a nucleic acid encodingLMNA which comprises a point mutation in exon 11. The point mutation canbe a C to T substitution in codon 608.

Further provided are methods of modulating splicing of LMNA pre-mRNA bycontacting cells or tissues with one or more of the antisense compounds.In one embodiment, the modulation of splicing results in an increase inthe ratio of full-length LMNA mRNA to truncated LMNA mRNA. In anotherembodiment, the modulation of splicing results in an increase in theratio of full-length lamin A protein to truncated lamin A protein.

Provided herein is a method for identifying cis splicing regulatoryelements of a selected pre-mRNA by selecting a splice site of thepre-mRNA, designing a set of antisense compounds targeting a region upto about 500, up to about 250, up to about 150, or up to about 75nucleotides 5′ (upstream) or 3′ (downstream) of the selected splice siteand screening the antisense compounds to identify compounds thatmodulate the ratio of splicing products of the pre-mRNA. The compoundsused in the methods provided herein do not elicit RNase H mediateddegradation of the target nucleic acid. These compounds are termed“RNase H-independent” compounds. In one embodiment, the cis regulatoryelement is a splicing silencer element. In another embodiment, the cisregulatory element is a splicing enhancer element. An increase in usageof the selected splice site upon binding of the antisense compoundindicates identification of a splicing silencer element and a decreasein usage of the selected splice site indicates identification of asplicing enhancer element.

The antisense compounds designed for identification of cis splicingregulatory elements can target splice acceptor sites, splice donorsites, constitutive splice sites, cryptic splice sites or aberrantsplice sites. The cis regulatory element can be in an intron or an exon.In some embodiments, the antisense compounds of the method are 12 to 30nucleobases in length. In other embodiments, the antisense compounds ofthe method are 16 to 20 nucleobases in length.

In one embodiment, the RNase H-independent compounds provided herein forthe method of identifying cis regulatory elements comprise at least onemodified nucleotide. In one embodiment, the antisense compounds comprisea modified nucleotide at each position. In a further embodiment, theantisense compounds comprise uniformly modified nucleotides. In oneembodiment, the modified nucleotide or nucleotides comprise a sugarmodification. In one aspect, the sugar modification is2′-O-methoxyethyl. In another aspect, the antisense compounds furthercomprise phosphorothioate linkages at each position. In one embodiment,the antisense compounds comprise at least one mimetic. In one aspect,the mimetic is a peptide nucleic acid. In another aspect, the mimetic isa morpholino group.

Also provided is the use of an antisense oligonucleotide targeted tohuman LMNA for the preparation of a medicament for the inhibition ofLMNA in a cell or tissue. Further provided is the use of a fullymodified antisense oligonucleotide for the preparation of a medicamentfor modulation of splicing of LMNA in a cell or tissue. The use of anantisense oligonucleotide targeted to human LMNA for the preparation ofa medicament for the treatment of HGPS is also provided.

DETAILED DESCRIPTION

Antisense technology is an effective means for reducing the expressionof one or more specific gene products and is uniquely useful in a numberof therapeutic, diagnostic, and research applications. Provided hereinare antisense compounds useful for modulating gene expression andassociated pathways via antisense mechanisms of action based on targetdegradation or target occupancy. Also provided herein are antisensecompounds useful for identification of cis splicing regulatory elementspresent in pre-mRNA molecules, including exonic splicing enhancers,exonic splicing silencers, intronic splicing enhancers and intronicsplicing silencers.

The principle behind antisense technology is that an antisense compound,which hybridizes to a target nucleic acid, modulates gene expressionactivities such as transcription, splicing or translation through one ofa number of antisense mechanisms. The sequence specificity of antisensecompounds makes them extremely attractive as tools for target validationand gene functionalization, as well as therapeutics to selectivelymodulate the expression of genes involved in disease.

The role of LMNA in diseases such as HGPS makes it an importanttherapeutic target. Both LMNA mRNA target knockdown and modulation ofLMNA splice product ratios are desirable outcomes. Thus, provided hereinare two types of antisense compounds targeting LMNA. One type ofantisense compound targets LMNA and results in target degradation. Thesecond type of compound targets specific components of LMNA pre-mRNA tomodulate splicing. The latter compounds do not elicit RNase H-dependentdegradation of the target (RNase H-independent compounds). Modulation ofsplicing is used to alter the ratio of splicing products to increase thedesired splice product and decrease the undesired splice product. In thecase of LMNA nucleic acids containing mutations in exon 11, which leadto development of HGPS, oligomeric compounds are designed to blocksplicing from a cryptic splice site activated by the mutation in exon11. In one aspect, the antisense compounds target splicing elements,such as splicing enhancer elements or splicing silencer elements. Theenhancer elements and silencer elements can found in introns or exons.

Processing of eukaryotic pre-mRNAs is a complex process that requires amultitude of signals and protein factors to achieve appropriate mRNAsplicing. Exon definition by the spliceosome requires more than thecanonical splicing signals which define intron-exon boundaries. One suchadditional signal is provided by cis-acting regulatory enhancer andsilencer sequences. Exonic splicing enhancers (ESE), exonic splicingsilencers (ESS), intronic splicing enhancers (ISE) and intron splicingsilencers (ISS) have been identified which either repress or enhanceusage of splice donor sites or splice acceptor sites, depending on theirsite and mode of action (Yeo et al. 2004, Proc. Natl. Acad. Sci. U.S.A.101(44):15700-15705). Binding of specific proteins (trans factors) tothese regulatory sequences directs the splicing process, eitherpromoting or inhibiting usage of particular splice sites and thusmodulating the ratio of splicing products (Scamborova et al. 2004, Mol.Cell. Biol. 24(5):1855-1869; Hovhannisyan and Carstens, 2005, Mol. Cell.Biol. 25(1):250-263; Minovitsky et al. 2005, Nucleic Acids Res.33(2):714-724). Little is known about the trans factors that interactwith intronic splicing elements; however, a number of studies haveprovided information on exonic splicing elements. For example, ESEs areknown to be involved in both alternative and constitutive splicing byacting as binding sites for members of the SR protein family. SRproteins bind to splicing elements via their RNA-binding domain andpromote splicing by recruiting spliceosomal components withprotein-protein interactions mediated by their RS domain, which iscomprised of several Arg-Ser dipeptides (Cartegni and Krainer, 2003,Nat. Struct. Biol. 10(2):120-125; Wang et al. 2005, Nucleic Acids Res.33(16):5053-5062). ESEs have been found to be enriched in regions ofexons that are close to splice sites, particularly 80 to 120 bases fromthe ends of splice acceptor sites (Wu et al. 2005, Genomics 86:329-336).Consensus sequences have been determined for four members of the SRprotein family, SF2/ASF, SC35, SRp40 and SRp55 (Cartegni et al. 2003,Nucleic Acids Res. 31(13):3568-3571). Although the trans factors thatbind intronic splicing regulatory elements have not been extensivelystudied, SR proteins and heterogeneous ribonucleoproteins (hnRNPs) haveboth been suggested to interact with these elements (Yeo et al. 2004,Proc. Natl. Acad. Sci. U.S.A. 101(44):15700-15705).

Identification of splicing enhancer and splicing silencer elements wouldbe of great value for identification of therapeutic tools useful formodulating splicing. Thus, provided herein are methods of identifyingsplicing elements using modified antisense compounds. It is shown hereinthat splicing elements can be identified by designing a series ofmodified antisense compounds targeting a region of a gene of interest,particularly targeting regions up to about 500 nucleotides upstream (5′)or downstream (3′) of a selected pre-mRNA splice site. Antisensecompounds also can be targeted to regions up to about 250, or up toabout 150, or up to about 75 nucleotides upstream or downstream of aselected pre-mRNA splice site. The splice sites can be splice donorsites (also known as 5′ splice sites) or splice acceptor sites (alsoknown as 3′ splice sites). Furthermore, the splice sites can beconstitutive (normal) splice sites or they can be cryptic or aberrantsplice sites. The modified antisense compounds are RNase H-independentand thus function by occupancy-based mechanisms. The antisense compoundsare then screened in an appropriate in vitro, ex vivo or in vivo systemto identify compounds that alter the ratio of splicing products of theselected pre-mRNA. Methods of screening antisense compounds are wellknown in the art. For example, an antisense compound targeting a regionupstream of a normal splice site which is found to enhance splicing ofan alternative transcript suggests the compound binds to a splicingenhancer element for the normal (constitutive) splice product.Alternatively, an antisense compound targeting a region upstream of anormal splice site which is found to enhance splicing of the normaltranscript suggests the compound binds to a splicing silencer elementfor the normal splice product. The same logic is applied to antisensecompounds targeting regions upstream of a cryptic, or alternative,splice site. The splicing regulatory elements can be in exons orintrons. While not wishing to be bound by theory, the modified antisensecompounds targeting splicing regulatory elements may function byblocking trails regulatory factors such as SR proteins or hnRNPs frombinding the splicing regulatory elements, which would alter recruitmentof spliceosomal components that regulate splice site selection andusage.

Identification of splicing elements for LMNA is illustrated herein;however, the methods provided herein can be used to identify cissplicing regulatory elements of any known pre-mRNA having at least onealternative splice product. The methods provided herein are particularlyuseful for identifying splicing regulatory elements for genes associatedwith genetic diseases, particularly those that result from alterationsin splicing. Up to 50% of point mutations linked to genetic diseases inhumans result in aberrant splicing. Such point mutations can altersplicing by directly inactivating or creating a splice site, indirectlyactivating a cryptic splice site or interfering with regulatory ciselements (Cartegni and Krainer, 2003, Nat. Struct. Biol. 10(2):120-125).Antisense oligonucleotides have been used to block cryptic splice sites,which were activated by mutations that inhibit use of the normal splicesites, for both human β-globin and CFTR (Lacerra et al. 2000, Proc.Natl. Acad. Sci. U.S.A. 97:9591-9596; Frieman et al. 1999, J. Biol.Chem. 274:36193-36199). Similarly, antisense oligonucleotides have beenused to modulate splicing of Bcl-x to favor either the short form or thelong form (Taylor et al. 1999, Nat. Biotechnol. 17:1097-1100) and toforce skipping of a specific dystrophin exon harboring a prematuretermination codon (Wilton et al. 1999, Neuromuscul. Disord. 9:330-338).

As used herein, the terms “target nucleic acid” and “nucleic acidmolecule encoding LMNA” have been used for convenience to encompass DNAencoding LMNA, RNA (including pre-mRNA and mRNA or portions thereof)transcribed from such DNA, and also cDNA derived from such RNA.

As used herein, “targeting” or “targeted to” refer to the process ofdesigning an oligomeric compound such that the compound hybridizes witha selected nucleic acid molecule or portion of a selected nucleic acidmolecule.

As used herein, “hybridization” means the pairing of complementarystrands of oligomeric compounds. In the context of the presentdisclosure, an oligomeric compound is specifically hybridizable whenthere is a sufficient degree of complementarity to avoid non-specificbinding of the oligomeric compound to non-target nucleic acid sequences.

As used herein, “antisense mechanisms” are all those involvinghybridization of a compound with target nucleic acid, wherein theoutcome or effect of the hybridization is either target degradation ortarget occupancy with concomitant stalling of the cellular machineryinvolving, for example, translation or splicing.

As used herein, “modulation of splicing” refers to altering theprocessing of a pre-mRNA transcript such that there is an increase ordecrease of one or more splice products, or a change in the ratio of twoor more splice products. Modulation of splicing can also refer toaltering the processing of a pre-mRNA transcript such that a splicedmRNA molecule contains either a different combination of exons as aresult of exon skipping or exon inclusion, a deletion in one or moreexons, or additional sequence not normally found in the spliced mRNA(e.g., intron sequence).

As used herein, “splice products” or “splicing products” are the maturemRNA molecules generated from the process of splicing a pre-mRNA.Alternatively spliced pre-mRNAs have at least two different spliceproducts. For example, a first splicing product may contain anadditional exon, or portion of an exon, relative to a second splicingproduct. Splice products of a selected pre-mRNA can be identified by avariety of different techniques well known to those of skill in the art.

As used herein, “splice site” refers to the junction between an exon andan intron in a pre-mRNA (unspliced RNA) molecule (also known as a“splice junction”). A “cryptic splice site” is a splice site that is nottypically used but may be used when the usual splice site is blocked orunavailable or when a mutation causes a normally dormant site to becomean active splice site. An “aberrant splice site” is a splice site thatresults from a mutation in the native DNA and mRNA. In the context ofthe present disclosure, an oligomeric compound “targeted to a splicesite” refers to a compound that hybridizes with at least a portion of aregion of nucleic acid encoding a splice site or a compound thathybridizes with an intron or exon in proximity to a splice site, suchthat splicing of the mRNA is modulated. In the context of the presentdisclosure, an antisense compound targeting a region up to about 500nucleobases upstream (5′) or downstream (3′) of a splice site refers toa compound that hybridizes with at least a portion of an intron or exonsequence that is 0 to 500 nucleobases upstream or downstream of thesplice site

As used herein “splice donor site” refers to a splice site found at the5′ end of an intron. Splice donor site is used interchangeably with “5′splice site.” As used herein “splice acceptor site” refers to a splicesite found at the 3′ end of an intron. Splice acceptor site is usedinterchangeably with “3′ splice site.”

As used herein, usage of a splice site refers to the cellularspliceosomal machinery recognizing and selecting a particular splicesite during the splicing process such that the spliced mRNA productreflects use of that site as either a splice donor site or a spliceacceptor site. In the context of the present disclosure, if usage of aparticular splice site is favored (such as by treatment with one of theantisense compounds provided herein) over a second splice site, theratio of mRNA products resulting from the splicing reaction will bealtered. One of skill in the art is able to determine the splice sitesof a selected pre-mRNA and predict the size and nature of the possiblesplicing products and thus determine which splice sites are being usedunder a given condition. For example, in the context of the presentdisclosure, an antisense compound that causes an increase in usage of aselected splice site indicates the compound is interfering with asplicing silencer element and an antisense compound that causes adecrease in usage of the selected splice site indicates the compound isinterfering with a splicing enhancer element. Thus, the antisensecompounds provided herein are useful for identifying the presence ofsplicing enhancer and splicing silencer elements by assessing the ratioof splicing products.

As used herein, “cis splicing regulatory elements” are regions ofsequence found in a pre-mRNA molecule which modulate splicing of themRNA. These regulatory elements are presumed to function throughinteraction with trans factors that either positively or negativelyregulate the cellular spliceosomal machinery. C is splicing regulatoryelements include splicing enhancers and splicing silencers. Splicingenhancers present in exons are termed “exonic splicing enhancers” or“ESEs” and splicing enhancers present in introns are termed “intronicsplicing enhancers” or “ISEs.” Similarly, splicing silencers present inexons are termed “exonic splicing silencers” or “ESSs” and splicingsilencers present in introns are termed “intronic splice silencers” or“ISSs.”

As used herein, “RNase H-independent” compounds are antisense compoundshaving a least one chemical modification such that when the compound isbound to a target nucleic acid, the modification increases resistance ofthe target nucleic acid to RNase H-mediated cleavage. Such modificationsinclude, but are not limited to, nucleotides with modified sugarmoieties. As used herein, nucleotides with modified sugar moietiesinclude, but are not limited to, any nucleotide wherein the2′-deoxyribose sugar has been substituted with a chemically modifiedsugar moiety. In the context of the present disclosure, chemicallymodified sugar moieties include, but are not limited to,2′-O-(2-methoxyethyl), 2′-fluoro, 2′-dimethylaminooxyethoxy,2′-dimethylaminoethoxyethoxy, 2′-guanidinium, 2′-O-guanidinium ethyl,2′-carbamate, 2′-aminooxy, 2′-acetamido, and bicyclic nucleic acids.Modified compounds that confer resistance to RNase H-mediated targetcleavage are thoroughly described herein and are well known in the art.

As used herein, “uniformly modified” refers to antisense compoundswherein each nucleotide comprises the same modification ormodifications.

As used herein, “Bicyclic nucleic acids” or “BNAs” refer to nucleicacids which have a modified ribofuranosyl moiety wherein two non-geminalring carbon atoms are bridged. Exemplary BNAs which are useful in thecontext of the present disclosure are BNAs which have a bridge betweenthe 4′ and the 2′ position of the ribofuranosyl moiety, such as, forexample, methyleneoxy (4′-CH2-O-2′) BNA and ethyleneoxy (4′-CH2CH2-O-2′)BNA.

Target Nucleic Acids

As used herein, “targeting” or “targeted to” refer to the process ofdesigning an oligomeric compound such that the compound hybridizes witha selected nucleic acid molecule. Targeting an oligomeric compound to aparticular target nucleic acid molecule can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose expression is to be modulated. As used herein, the terms “targetnucleic acid” and “nucleic acid encoding LMNA” encompass DNA encodingLMNA, RNA (including pre-mRNA and mRNA) transcribed from such DNA, andalso cDNA derived from such RNA. As disclosed herein, the target nucleicacid encodes LMNA.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect (e.g.,modulation of expression) will result. “Region” is defined as a portionof the target nucleic acid having at least one identifiable structure,function, or characteristic. Target regions may include, for example, aparticular exon or intron, or may include only selected nucleobaseswithin an exon or intron which are identified as appropriate targetregions. Within regions of target nucleic acids are segments. “Segments”are defined as smaller or sub-portions of regions within a targetnucleic acid. “Sites,” as used herein, are defined as unique nucleobasepositions within a target nucleic acid. As used herein, the “targetsite” of an oligomeric compound is the 5′-most nucleotide of the targetnucleic acid to which the compound binds.

Provided herein are compositions and methods for modulating theexpression of LMNA (also known as EMD2; FPL; FPLD; LDP1; LFP; LMN1;lamin A; lamin A/C; lamin C; nuclear envelope protein lamin A precursor;nuclear envelope protein lamin C precursor). Listed in Table 1 areGENBANK® accession numbers of sequences used to design antisensecompounds targeted to LMNA. Antisense compounds include compounds whichhybridize with one or more target nucleic acid molecules shown in Table1, as well as antisense compounds which hybridize to other nucleic acidmolecules encoding LMNA. The antisense compounds may target any region,segment, or site of nucleic acid molecules which encode LMNA. Suitabletarget regions, segments, and sites include, but are not limited to, the5′UTR, the start codon, the stop codon, the coding region, the 3′UTR,the 5′ cap region, introns, exons, intron-exon junctions, exon-intronjunctions, exon-exon junctions, splice sites and splicing regulatoryelements.

TABLE 1 LMNA Target Sequences Target SEQ ID Name Species Genbank ® # NOLMNA Human AY357727.1 1 LMNA Human BC014507.1 2 LMNA Human NM_005572.2 3LMNA Human NM_170707.1 4 LMNA Human NM_170708.1 5 LMNA Human NM_170717.16 LMNA Human nucleotides 2533930 to 2560103 of 7 NT_079484.1Modulation of Target Expression

Modulation of expression of a target nucleic acid can be achievedthrough alteration of any number of nucleic acid (DNA or RNA) functions.“Modulation” means a perturbation of function, for example, either anincrease (stimulation or induction) or a decrease (inhibition orreduction) in expression. As another example, modulation of expressioncan include perturbing splice site selection of pre-mRNA processing.“Expression” includes all the functions by which a gene's codedinformation is converted into structures present and operating in acell. These structures include the products of transcription andtranslation. “Modulation of expression” means the perturbation of suchfunctions. The functions of DNA to be modulated can include replicationand transcription. Replication and transcription, for example, can befrom an endogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be modulated can includetranslocation functions, which include, but are not limited to,translocation of the RNA to a site of protein translation, translocationof the RNA to sites within the cell which are distant from the site ofRNA synthesis, and translation of protein from the RNA. RNA processingfunctions that can be modulated include, but are not limited to,splicing of the RNA to yield one or more RNA species, capping of theRNA, 3′ maturation of the RNA and catalytic activity or complexformation involving the RNA which may be engaged in or facilitated bythe RNA. Modulation of expression can result in the increased level ofone or more nucleic acid species or the decreased level of one or morenucleic acid species, either temporally or by net steady state level.One result of such interference with target nucleic acid function ismodulation of the expression of LMNA. Thus, in one embodiment modulationof expression can mean increase or decrease in target RNA or proteinlevels. In another embodiment modulation of expression can mean anincrease or decrease of one or more RNA splice products, or a change inthe ratio of two or more splice products.

Modulation of LMNA expression can be assayed in a variety of ways knownin the art. LMNA mRNA levels can be quantitated by, e.g., Northern blotanalysis, competitive polymerase chain reaction (PCR), or real-time PCR.RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA bymethods known in the art. Levels of a protein encoded by LMNA can bequantitated in a variety of ways well known in the art, such asimmunoprecipitation, Western blot analysis (immunoblotting), ELISA orfluorescence-activated cell sorting (FACS).

Kits, Research Reagents and Diagnostics

The antisense compounds provided herein can be utilized for diagnostics,and as research reagents and kits. Furthermore, antisense compounds,which are able to inhibit gene expression or modulate gene expressionwith specificity, are often used by those of ordinary skill to elucidatethe function of particular genes or to distinguish between functions ofvarious members of a biological pathway.

For use in kits and diagnostics, the antisense compounds providedherein, either alone or in combination with other compounds ortherapeutics, can be used as tools in differential and/or combinatorialanalyses to elucidate expression patterns of a portion or the entirecomplement of genes expressed within cells and tissues. Methods of geneexpression analysis are well known to those skilled in the art.

Therapeutics

Antisense compounds provided herein can be used to modulate theexpression of LMNA in an animal, such as a human. In one non-limitingembodiment, the methods comprise the step of administering to saidanimal in need of therapy for a disease or condition associated withLMNA an effective amount of an antisense compound that modulatesexpression of LMNA. A disease or condition associated with LMNAincludes, but is not limited to, HGPS. Antisense compounds thateffectively modulate expression of L/NA RNA or protein products ofexpression are considered active antisense compounds.

For example, modulation of expression of LMNA can be measured in abodily fluid, which may or may not contain cells; tissue; or organ ofthe animal. Methods of obtaining samples for analysis, such as bodyfluids (e.g., sputum, serum), tissues (e.g., biopsy), or organs, andmethods of preparation of the samples to allow for analysis are wellknown to those skilled in the art. Methods for analysis of RNA andprotein levels are discussed above and are well known to those skilledin the art. The effects of treatment can be assessed by measuringbiomarkers associated with the target gene expression in theaforementioned fluids, tissues or organs, collected from an animalcontacted with one or more compounds, by routine clinical methods knownin the art. These biomarkers include but are not limited to: livertransaminases, bilirubin, albumin, blood urea nitrogen, creatine andother markers of kidney and liver function; interleukins, tumor necrosisfactors, intracellular adhesion molecules, C-reactive protein,chemokines, cytokines, and other markers of inflammation.

The antisense compounds provided herein can be utilized inpharmaceutical compositions by adding an effective amount of a compoundto a suitable pharmaceutically acceptable diluent or carrier. Acceptablecarriers and diluents are well known to those skilled in the art.Selection of a diluent or carrier is based on a number of factors,including, but not limited to, the solubility of the compound and theroute of administration. Such considerations are well understood bythose skilled in the art. The compounds provided herein can also be usedin the manufacture of a medicament for the treatment of diseases anddisorders related to LMNA.

Methods whereby bodily fluids, organs or tissues are contacted with aneffective amount of one or more of the antisense compounds orcompositions are also contemplated. Bodily fluids, organs or tissues canbe contacted with one or more of the compounds described hereinresulting in modulation of LMNA expression in the cells of bodilyfluids, organs or tissues. An effective amount can be determined bymonitoring the modulatory effect of the antisense compound or compoundsor compositions on target nucleic acids or their products by methodsroutine in the art.

Thus, provided herein is the use of an antisense compound targeted toLMNA in the manufacture of a medicament for the treatment of a diseaseor disorder by means of the method described above.

Antisense Compounds

The term “oligomeric compound” refers to a polymeric structure capableof hybridizing to a region of a nucleic acid molecule. This termincludes oligonucleotides, oligonucleosides, oligonucleotide analogs,oligonucleotide mimetics and chimeric combinations of these. Oligomericcompounds are routinely prepared linearly but can be joined or otherwiseprepared to be circular. Moreover, branched structures are known in theart. An “antisense compound” or “antisense oligomeric compound” refersto an oligomeric compound that is at least partially complementary tothe region of a nucleic acid molecule to which it hybridizes and whichmodulates its expression. Consequently, while all antisense compoundscan be said to be oligomeric compounds, not all oligomeric compounds areantisense compounds. An “antisense oligonucleotide” is an antisensecompound that is a nucleic acid-based oligomer. An antisenseoligonucleotide can be chemically modified. Nonlimiting examples ofoligomeric compounds include primers, probes, antisense compounds,antisense oligonucleotides, external guide sequence (EGS)oligonucleotides and alternate splicers. In one embodiment, theoligomeric compound comprises an antisense strand hybridized to a sensestrand. Oligomeric compounds can be introduced in the form ofsingle-stranded, double-stranded, circular, branched or hairpins and cancontain structural elements such as internal or terminal bulges orloops. Oligomeric double-stranded compounds can be two strandshybridized to form double-stranded compounds or a single strand withsufficient self complementarity to allow for hybridization and formationof a fully or partially double-stranded compound.

The oligomeric compounds comprise compounds from 8 to 80 nucleobases(i.e. from 8 to 80 linked nucleosides). One will appreciate that thiscomprehends antisense compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases.

In one embodiment, the antisense compounds comprise 13 to 80nucleobases. One will appreciate that this embodies antisense compoundsof 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 2930, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80nucleobases.

In one embodiment, the antisense compounds comprise 12 to 50nucleobases. One will appreciate that this embodies antisense compoundsof 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, or 50 nucleobases.

In one embodiment, the antisense compounds comprise 12 to 30nucleobases. One will appreciate that this embodies antisense compoundsof 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29 or 30 nucleobases.

In some embodiments, the antisense compounds comprise 15 to 30nucleobases. One will appreciate that this embodies antisense compoundsof 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nucleobases.

In one embodiment, the antisense compounds comprise 20 to 30nucleobases. One will appreciate that this embodies antisense compoundsof 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases.

In one embodiment, the antisense compounds comprise 20 to 24nucleobases. One will appreciate that this embodies antisense compoundsof 20, 21, 22, 23, or 24 nucleobases.

In one embodiment, the antisense compounds comprise 16 to 20nucleobases. One will appreciate that this embodies antisense compoundsof 16, 17, 18, 19 or 20 nucleobases.

In one embodiment, the antisense compounds comprise 20 nucleobases.

In one embodiment, the antisense compounds comprise 19 nucleobases.

In one embodiment, the antisense compounds comprise 18 nucleobases.

In one embodiment, the antisense compounds comprise 17 nucleobases.

In one embodiment, the antisense compounds comprise 16 nucleobases.

In one embodiment, the antisense compounds comprise 15 nucleobases.

In one embodiment, the antisense compounds comprise 14 nucleobases.

In one embodiment, the antisense compounds comprise 13 nucleobases.

Antisense compounds 8-80 nucleobases in length, or any length within therecited range, comprising a stretch of at least eight (8) consecutivenucleobases selected from within the illustrative antisense compoundsare considered to be suitable antisense compounds.

Compounds provided herein include oligonucleotide sequences thatcomprise at least the 8 consecutive nucleobases from the 5′-terminus ofone of the illustrative antisense compounds (the remaining nucleobasesbeing a consecutive stretch of the same oligonucleotide beginningimmediately upstream of the 5′-terminus of the antisense compound whichis specifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains 8 to 80, or 13 to 80, or 12 to 50, or12 to 30, or 15 to 30, or 20 to 30, or 20 to 24, or 16 to 20nucleobases). Other compounds are represented by oligonucleotidesequences that comprise at least the 8 consecutive nucleobases from the3′-terminus of one of the illustrative antisense compounds (theremaining nucleobases being a consecutive stretch of the sameoligonucleotide beginning immediately downstream of the 3′-terminus ofthe antisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the oligonucleotide contains about 8to about 80, or about 13 to about 80, or about 12 to about 50, or about12 to about 30, or about 15 to about 30, or about 20 to about 30, orabout 20 to about 24, or about 16 to about 20 nucleobases). It is alsounderstood that compounds may be represented by oligonucleotidesequences that comprise at least 8 consecutive nucleobases from aninternal portion of the sequence of an illustrative compound, and mayextend in either or both directions until the oligonucleotide contains 8to 80, or 13 to 80, or 12 to 50, or 12 to 30, or 15 to 30, or 20 to 30,or 20 to 24, or 16 to 20 nucleobases.

Validated Target Segments

The locations on the target nucleic acid to which active oligomericcompounds hybridize are herein below referred to as “validated targetsegments.” As used herein the term “validated target segment” is definedas at least an 8-nucleobase portion (i.e. 8 consecutive nucleobases) ofa target region to which an active oligomeric compound is targeted.While not wishing to be bound by theory, it is presently believed thatthese target segments represent portions of the target nucleic acidwhich are accessible for hybridization.

Target segments can include DNA or RNA sequences that comprise at leastthe 8 consecutive nucleobases from the 5′-terminus of a validated targetsegment (the remaining nucleobases being a consecutive stretch of thesame DNA or RNA beginning immediately upstream of the 5′-terminus of thetarget segment and continuing until the DNA or RNA contains about 8 toabout 80 nucleobases). Similarly validated target segments arerepresented by DNA or RNA sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of a validated targetsegment (the remaining nucleobases being a consecutive stretch of thesame DNA or RNA beginning immediately downstream of the 3′-terminus ofthe target segment and continuing until the DNA or RNA contains about 8to about 80 nucleobases). It is also understood that a validatedoligomeric target segment can be represented by DNA or RNA sequencesthat comprise at least 8 consecutive nucleobases from an internalportion of the sequence of a validated target segment, and can extend ineither or both directions until the oligonucleotide contains about 8 toabout 80 nucleobases.

The validated target segments identified herein can be employed in ascreen for additional compounds that modulate the expression of LMNA.“Modulators” are those compounds that modulate the expression of LMNAand which comprise at least an 8-nucleobase portion (i.e. 8 consecutivenucleobases) which is complementary to a validated target segment. Thescreening method comprises the steps of contacting a validated targetsegment of a nucleic acid molecule encoding LMNA with one or morecandidate modulators, and selecting for one or more candidate modulatorswhich perturb the expression of a nucleic acid molecule encoding LMNA.Once it is shown that the candidate modulator or modulators are capableof modulating the expression of a nucleic acid molecule encoding LMNA,the modulator can then be employed in further investigative studies ofthe function of LMNA, or for use as a research, diagnostic, ortherapeutic agent. Modulator compounds of LMNA can also be identified orfurther investigated using one or more phenotypic assays, each havingmeasurable endpoints predictive of efficacy in the treatment of aparticular disease state or condition. Phenotypic assays, kits andreagents for their use are well known to those skilled in the art.

Hybridization

“Hybridization” means the pairing of complementary strands of oligomericcompounds. While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases) of thestrands of oligomeric compounds. For example, adenine and thymine arecomplementary nucleobases which pair through the formation of hydrogenbonds, Hybridization can occur under varying circumstances.

An oligomeric compound is specifically hybridizable when there is asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

“Stringent hybridization conditions” or “stringent conditions” refer toconditions under which an oligomeric compound will hybridize to itstarget sequence, but to a minimal number of other sequences. Stringentconditions are sequence-dependent and will be different in differentcircumstances, and “stringent conditions” under which oligomericcompounds hybridize to a target sequence are determined by the natureand composition of the oligomeric compounds and the assays in which theyare being investigated.

Complementarity

“Complementarity,” as used herein, refers to the capacity for precisepairing between two nucleobases on one or two oligomeric compoundstrands. For example, if a nucleobase at a certain position of anantisense compound is capable of hydrogen bonding with a nucleobase at acertain position of a target nucleic acid, then the position of hydrogenbonding between the oligonucleotide and the target nucleic acid isconsidered to be a complementary position. The oligomeric compound andthe further DNA or RNA are complementary to each other when a sufficientnumber of complementary positions in each molecule are occupied bynucleobases which can hydrogen bond with each other. Thus, “specificallyhybridizable” and “complementary” are terms which are used to indicate asufficient degree of precise pairing or complementarity over asufficient number of nucleobases such that stable and specific bindingoccurs between the oligomeric compound and a target nucleic acid.

It is understood in the art that the sequence of an oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure,mismatch or hairpin structure). The oligomeric compounds provided hereincomprise at least 70%, or at least 75%, or at least 80%, or at least85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%,or at least 98%, or at least 99% sequence complementarity to a targetnucleic acid sequence. For example, an oligomeric compound in which 18of 20 nucleobases of the antisense compound are complementary to atarget nucleic acid, and would therefore specifically hybridize, wouldrepresent 90 percent complementarity. In this example, the remainingnoncomplementary nucleobases may be clustered or interspersed withcomplementary nucleobases and need not be contiguous to each other or tocomplementary nucleobases. As such, an oligomeric compound which is 18nucleobases in length having 4 (four) noncomplementary nucleobases whichare flanked by two regions of complete complementarity with the targetnucleic acid would have 77.8% overall complementarity with the targetnucleic acid and would thus fall within the scope of the compoundsprovided herein. Percent complementarity of an oligomeric compound witha region of a target nucleic acid can be determined routinely usingBLAST programs (basic local alignment search tools) and PowerBLASTprograms known in the art (Altschul et al., J. Mol. Biol., 1990, 215,403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). Percenthomology, sequence identity or complementarity, can be determined by,for example, the Gap program (Wisconsin Sequence Analysis Package,Version 8 for Unix, Genetics Computer Group, University Research Park,Madison Wis.), using default settings, which uses the algorithm of Smithand Waterman (Adv. Appl. Math., 1981, 2, 482-489).

Identity

Antisense compounds, or a portion thereof, may have a defined percentidentity to a SEQ ID NO, or a compound having a specific Isis number. Asused herein, a sequence is identical to the sequence disclosed herein ifit has the same nucleobase pairing ability. For example, a RNA whichcontains uracil in place of thymidine in the disclosed sequences wouldbe considered identical as they both pair with adenine. This identitymay be over the entire length of the oligomeric compound, or in aportion of the antisense compound (e.g., nucleobases 1-20 of a 27-mermay be compared to a 20-mer to determine percent identity of theoligomeric compound to the SEQ ID NO.) It is understood by those skilledin the art that an antisense compound need not have an identicalsequence to those described herein to function similarly to theantisense compound described herein. Shortened versions of antisensecompound taught herein, or non-identical versions of the antisensecompound taught herein are also contemplated. Non-identical versions arethose wherein each base does not have the same pairing activity as theantisense compounds disclosed herein. Bases do not have the same pairingactivity by being shorter or having at least one abasic site.Alternatively, a non-identical version can include at least one basereplaced with a different base with different pairing activity (e.g., Gcan be replaced by C, A, or T). Percent identity is calculated accordingto the number of bases that have identical base pairing corresponding tothe SEQ ID NO or antisense compound to which it is being compared. Thenon-identical bases may be adjacent to each other, dispersed through outthe oligonucleotide, or both.

For example, a 16-mer having the same sequence as nucleobases 2-17 of a20-mer is 80% identical to the 20-mer. Alternatively, a 20-mercontaining four nucleobases not identical to the 20-mer is also 80%identical to the 20-mer. A 14-mer having the same sequence asnucleobases 1-14 of an 18-mer is 78% identical to the 18-mer. Suchcalculations are well within the ability of those skilled in the art.

The percent identity is based on the percent of nucleobases in theoriginal, or first, sequence present in a portion of the modified, orsecond, sequence. In a preferred embodiment, the oligonucleotides are atleast about 80%, at least about 85%, at least about 90%, at least about95% or 100% identical to at least a portion of one of the illustratedantisense compounds, or of the complement of the active target segmentspresented herein.

It is well known by those skilled in the art that it is possible toincrease or decrease the length of an antisense compound and/orintroduce mismatch bases without eliminating activity. For example, inWoolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992,incorporated herein by reference), a series of ASOs 13-25 nucleobases inlength were tested for their ability to induce cleavage of a target RNA.ASOs 25 nucleobases in length with 8 or 11 mismatch bases near the endsof the ASOs were able to direct specific cleavage of the target mRNA,albeit to a lesser extent than the ASOs that contained no mismatches.Similarly, target specific cleavage was achieved using a 13 nucleobaseASOs, including those with 1 or 3 mismatches. Maher and Dolnick (Nuc.Acid. Res. 16:3341-3358, 1988, incorporated herein by reference) testeda series of tandem 14 nucleobase ASOs, and a 28 and 42 nucleobase ASOscomprised of the sequence of two or three of the tandem ASOs,respectively, for their ability to arrest translation of human DHFR in arabbit reticulocyte assay. Each of the three 14 nucleobase ASOs alonewere able to inhibit translation, albeit at a more modest level than the28 or 42 nucleobase ASOs. It is understood that antisense compounds canvary in length and percent complementarity to the target provided thatthey maintain the desired activity. Methods to determine desiredactivity are disclosed herein and well known to those skilled in theart.

Chemical Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base(sometimes referred to as a “nucleobase” or simply a “base”). The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety ofthe sugar. In forming oligonucleotides, the phosphate groups covalentlylink adjacent nucleosides to one another to form a linear polymericcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage. It is often preferable to include chemicalmodifications in oligonucleotides to alter their activity. Chemicalmodifications can alter oligonucleotide activity by, for example:increasing affinity of an antisense oligonucleotide for its target RNA,increasing nuclease resistance, and/or altering the pharmacokinetics ofthe oligonucleotide. The use of chemistries that increase the affinityof an oligonucleotide for its target can allow for the use of shorteroligonucleotide compounds.

The term “nucleobase” or “heterocyclic base moiety” as used herein,refers to the heterocyclic base portion of a nucleoside. In general, anucleobase is any group that contains one or more atom or groups ofatoms capable of hydrogen bonding to a base of another nucleic acid. Inaddition to “unmodified” or “natural” nucleobases such as the purinenucleobases adenine (A) and guanine (G), and the pyrimidine nucleobasesthymine (T), cytosine (C) and uracil (U), many modified nucleobases ornucleobase mimetics known to those skilled in the art are amenable tothe compounds described herein. The terms modified nucleobase andnucleobase mimetic can overlap but generally a modified nucleobaserefers to a nucleobase that is fairly similar in structure to the parentnucleobase, such as for example a 7-deaza purine, a 5-methyl cytosine,or a G-clamp, whereas a nucleobase mimetic would include morecomplicated structures, such as for example a tricyclic phenoxazinenucleobase mimetic. Methods for preparation of the above noted modifiednucleobases are well known to those skilled in the art.

Antisense compounds provided herein may also contain one or morenucleosides having modified sugar moieties. The furanosyl sugar ring ofa nucleoside can be modified in a number of ways including, but notlimited to, addition of a substituent group, bridging of two non-geminalring atoms to form a bicyclic nucleic acid (BNA) and substitution of anatom or group such as —S—, —N(R)— or —C(R₁)(R₂) for the ring oxygen atthe 4′-position. Modified sugar moieties are well known and can be usedto alter, typically increase, the affinity of the antisense compound forits target and/or increase nuclease resistance. A representative list ofmodified sugars includes but is not limited to bicyclic modified sugars(BNA's), including methyleneoxy (4′-CH2-O-2′) BNA and ethyleneoxy(4′-CH2CH2-O-2′) BNA; and substituted sugars, especially 2′-substitutedsugars having a 2′-F, 2′-OCH₂ or a 2′-O(CH₂)₂—OCH₃ substituent group.Sugars can also be replaced with sugar mimetic groups among others.Methods for the preparations of modified sugars are well known to thoseskilled in the alt. In the context of the present disclosure, chemicallymodified sugar moieties include, but are not limited to,2′-O-(2-methoxyethyl), 2′-fluoro, 2′-dimethylaminooxyethoxy,2′-dimethylaminoethoxyethoxy, 2′-guanidinium, 2′-O-guanidinium ethyl,2′-carbamate, 2′-aminooxy, 2′-acetamido, methyleneoxy (4′-CH2-O-2′) BNAand ethyleneoxy (4′-CH2CH2-O-2′) BNA.

The compounds described herein may include internucleoside linkinggroups that link the nucleosides or otherwise modified monomer unitstogether thereby forming an antisense compound. The two main classes ofinternucleoside linking groups are defined by the presence or absence ofa phosphorus atom. Representative phosphorus containing internucleosidelinkages include, but are not limited to, phosphodiesters,phosphotriesters, methylphosphonates, phosphoramidate, andphosphorothioates. Representative non-phosphorus containinginternucleoside linking groups include, but are not limited to,methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—), thiodiester (—O—C(O)—S—),thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H)2-O—); andN,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—). Antisense compounds havingnon-phosphorus internucleoside linking groups are referred to asoligonucleosides. Modified internucleoside linkages, compared to naturalphosphodiester linkages, can be used to alter, typically increase,nuclease resistance of the antisense compound. Internucleoside linkageshaving a chiral atom can be prepared racemic, chiral, or as a mixture.Representative chiral internucleoside link-ages include, but are notlimited to, alkylphosphonates and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinglinkages are well known to those skilled in the art.

As used herein the term “mimetic” refers to groups that are substitutedfor a sugar, a nucleobase, and/or internucleoside linkage. Generally, amimetic is used in place of the sugar or sugar-internucleoside linkagecombination, and the nucleobase is maintained for hybridization to aselected target. Representative examples of a sugar mimetic include, butare not limited to, cyclohexenyl or morpholino. Representative examplesof a mimetic for a sugar-internucleoside link-age combination include,but are not limited to, peptide nucleic acids (PNA) and morpholinogroups linked by uncharged achiral linkages. In some instances a mimeticis used in place of the nucleobase. Representative nucleobase mimeticsare well known in the art and include, but are not limited to, tricyclicphenoxazine analogs and universal bases (Berger et al., Nuc Acid Res.2000, 28:2911-14, incorporated herein by reference). Methods ofsynthesis of sugar, nucleoside and nucleobase mimetics are well known tothose skilled in the art.

As used herein the term “nucleoside” includes, nucleosides, abasicnucleosides, modified nucleosides, and nucleosides having mimetic basesand/or sugar groups.

As used herein, the term “oligonucleotide” refers to an oligomericcompound which is an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA). This term includes oligonucleotidescomposed of naturally- and non-naturally-occurring nucleobases, sugarsand covalent internucleoside linkages, possibly further includingnon-nucleic acid conjugates.

The present disclosure provides compounds having reactive phosphorusgroups useful for forming internucleoside linkages including for examplephosphodiester and phosphorothioate internucleoside linkages. Methods ofpreparation and/or purification of precursors or antisense compounds arenot a limitation of the compositions or methods provided herein. Methodsfor synthesis and purification of DNA, RNA, and the antisense compoundsprovided herein are well known to those skilled in the art.

As used herein the term “chimeric antisense compound” refers to anantisense compound, having at least one sugar, nucleobase and/orinternucleoside linkage that is differentially modified as compared tothe other sugars, nucleobases and internucleoside linkages within thesame oligomeric compound. The remainder of the sugars, nucleobases andinternucleoside linkages can be independently modified or unmodified. Ingeneral a chimeric oligomeric compound will have modified nucleosidesthat can be in isolated positions or grouped together in regions thatwill define a particular motif. Any combination of modifications and ormimetic groups can comprise a chimeric oligomeric compound.

Chimeric oligomeric compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligomeric compound mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease thatcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of inhibition of gene expression. Consequently,comparable results can often be obtained with shorter oligomericcompounds when chimeras are used, compared to for examplephosphorothioate deoxyoligonucleotides hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

As used herein, the term “fully modified motif” refers to an antisensecompound comprising a contiguous sequence of nucleosides whereinessentially each nucleoside is a sugar modified nucleoside havinguniform modification.

The compounds described herein contain one or more asymmetric centersand thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), a or β, or as (D) or (L) such as foramino acids et al. The present disclosure is meant to include all suchpossible isomers, as well as their racemic and optically pure forms.

In one aspect, antisense compounds are modified by covalent attachmentof one or more conjugate groups. Conjugate groups may be attached byreversible or irreversible attachments. Conjugate groups may be attacheddirectly to antisense compounds or by use of a linker. Linkers may bemono- or bifunctional linkers. Such attachment methods and linkers arewell known to those skilled in the art. In general, conjugate groups areattached to antisense compounds to modify one or more properties. Suchconsiderations, are well known to those skilled in the art.

Oligomer Synthesis

Oligomerization of modified and unmodified nucleosides can be routinelyperformed according to literature procedures for DNA (Protocols forOligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/orRNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications ofChemically synthesized RNA in RNA: Protein Interactions, Ed. Smith(1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).

Antisense compounds can be conveniently and routinely made through thewell-known technique of solid phase synthesis. Equipment for suchsynthesis is sold by several vendors including, for example, AppliedBiosystems (Foster City, Calif.). Any other means for such synthesisknown in the art may additionally or alternatively be employed. It iswell known to use similar techniques to prepare oligonucleotides such asthe phosphorothioates and alkylated derivatives. The disclosure is notlimited by the method of antisense compound synthesis.

Oligomer Purification and Analysis

Methods of oligonucleotide purification and analysis are known in theart. Analysis methods include capillary electrophoresis (CE) andelectrospray-mass spectroscopy. Such synthesis and analysis methods canbe performed in multi-well plates. The methods described herein are notlimited by the method of oligomer purification.

Salts, Prodrugs and Bioequivalents

The antisense compounds described herein comprise any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any otherfunctional chemical equivalent which, upon administration to an animalincluding a human, is capable of providing (directly or indirectly) thebiologically active metabolite or residue thereof. Accordingly, forexample, the disclosure is also drawn to prodrugs and pharmaceuticallyacceptable salts of the antisense compounds, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive or less active form that is converted to an active form (i.e.,drug) within the body or cells thereof by the action of endogenousenzymes, chemicals, and/or conditions. In particular, prodrug versionsof the oligonucleotides are prepared as SATE ((S-acetyl-2-thioethyl)phosphate) derivatives according to the methods disclosed in WO 93/24510or WO 94/26764. Prodrugs can also include antisense compounds whereinone or both ends comprise nucleobases that are cleaved (e.g., byincorporating phosphodiester backbone linkages at the ends) to producethe active compound.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds: i.e., salts thatretain the desired biological activity of the parent compound and do notimpart undesired toxicological effects thereto. Sodium salts ofantisense oligonucleotides are useful and are well accepted fortherapeutic administration to humans. In another embodiment, sodiumsalts of dsRNA compounds are also provided.

Formulations

The antisense compounds described herein may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds.

The present disclosure also includes pharmaceutical compositions andformulations which include the antisense compounds described herein. Thepharmaceutical compositions may be administered in a number of waysdepending upon whether local or systemic treatment is desired and uponthe area to be treated.

The pharmaceutical formulations, which may conveniently be presented inunit dosage form, may be prepared according to conventional techniqueswell known in the pharmaceutical industry. Such techniques include thestep of bringing into association the active ingredients with thepharmaceutical carrier(s) or excipient(s). In general, the formulationsare prepared by uniformly and intimately bringing into association theactive ingredients with liquid carriers, finely divided solid carriers,or both, and then, if necessary, shaping the product (e.g., into aspecific particle size for delivery). In a preferred embodiment, thepharmaceutical formulations are prepared for pulmonary administration inan appropriate solvent, e.g., water or normal saline, possibly in asterile formulation, with carriers or other agents to allow for theformation of droplets of the desired diameter for delivery usinginhalers, nasal delivery devices, nebulizers, and other devices forpulmonary delivery. Alternatively, the pharmaceutical formulations maybe formulated as dry powders for use in dry powder inhalers.

A “pharmaceutical carrier” or “excipient” can be a pharmaceuticallyacceptable solvent, suspending agent or any other pharmacologicallyinert vehicle for delivering one or more nucleic acids to an animal andare known in the art. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition.

Combinations

Compositions provided herein can contain two or more antisensecompounds. In another related embodiment, compositions can contain oneor more antisense compounds, particularly oligonucleotides, targeted toa first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Alternatively, compositionsprovided herein can contain two or more antisense compounds targeted todifferent regions of the same nucleic acid target. Two or more combinedcompounds may be used together or sequentially. Compositions can also becombined with other non-antisense compound therapeutic agents.

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods have been describedherein with specificity in accordance with certain embodiments, thefollowing examples serve only to illustrate the disclosed compounds andare not intended to limit the same. Each of the references, GENBANK®accession numbers, and the like recited in the present application isincorporated herein by reference in its entirety.

EXAMPLE 1 Cell Culture and Treatment with Oligomeric Compounds

The effect of oligomeric compounds on target nucleic acid expression wastested in A549 or T24 cells. The human lung carcinoma cell line A549 wasobtained from the American Type Culture Collection (Manassas, Va.). A549cells are routinely cultured in DMEM, high glucose (Invitrogen LifeTechnologies, Carlsbad, Calif.) supplemented with 10% fetal bovineserum, 100 units per ml penicillin, and 100 micrograms per mlstreptomycin (Invitrogen Life Technologies, Carlsbad, Calif.). Cells areroutinely passaged by trypsinization and dilution when they reachedapproximately 90% confluence. Cells are seeded into 96-well plates(Falcon-Primaria #3872) at a density of approximately 5000 cells/wellfor use in oligomeric compound transfection experiments.

The transitional cell bladder carcinoma cell line T24 was obtained fromthe American Type Culture Collection (ATCC) (Manassas, Va.). T24 cellsare routinely cultured in complete McCoy's 5A basal media (InvitrogenLife Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovineserum (Invitrogen Life Technologies, Carlsbad, Calif.), penicillin 100units per mL, and streptomycin 100 μg/mL (Invitrogen Life Technologies,Carlsbad, Calif.). Cells are routinely passaged by trypsinization anddilution when they reach 90% confluence. Cells are seeded into 96-wellplates (Falcon-Primaria #3872) at a density of 7000 cells/well fortreatment with the antisense compound.

When cells reach appropriate confluency, they are treated witholigonucleotide using Lipofectin™ as described. When cells reached65-75% confluency, they were treated with oligonucleotide.Oligonucleotide was mixed with LIPOFECTIN™ Invitrogen Life Technologies,Carlsbad, Calif.) in Opti-MEM™-1 reduced serum medium (Invitrogen LifeTechnologies, Carlsbad, Calif.) to achieve the desired concentration ofoligonucleotide and a LIPOFECTIN™ concentration of 2.5 or 3 μg/mL per100 nM oligonucleotide. This transfection mixture was incubated at roomtemperature for approximately 0.5 hours. For cells grown in 96-wellplates, wells were washed once with 100 μL OPTI-MEM™-1 and then treatedwith 130 μL of the transfection mixture. Cells grown in 24-well platesor other standard tissue culture plates are treated similarly, usingappropriate volumes of medium and oligonucleotide. Cells are treated anddata are obtained in duplicate or triplicate. After approximately 4-7hours of treatment at 37° C., the medium containing the transfectionmixture was replaced with fresh culture medium. Cells were harvested16-24 hours after oligonucleotide treatment.

Control oligonucleotides are used to determine the optimal oligomericcompound concentration for a particular cell line. Furthermore, whenoligomeric compounds are tested in oligomeric compound screeningexperiments or phenotypic assays, control oligonucleotides are tested inparallel. The concentration of oligonucleotide used varies from cellline to cell line.

EXAMPLE 2 Real-Time Quantitative PCR Analysis of LMNA mRNA Levels

Quantitation of LMNA mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured were evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. After isolation theRNA is subjected to sequential reverse transcriptase (RT) reaction andreal-time PCR, both of which are performed in the same well. RT and PCRreagents were obtained from Invitrogen Life Technologies (Carlsbad,Calif.). RT, real-time PCR was carried out in the same by adding 20 μLPCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each ofdATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverseprimer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM®Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-wellplates containing 30 μL total RNA solution (20-200 ng). The RT reactionwas carried out by incubation for 30 minutes at 48° C. Following a 10minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles ofa two-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by RT, real-time PCR were normalizedusing either the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression was quantified by RT,real-time PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA was quantified using RiboGreen™RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).

170 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350in 10 n mM Tris-HCl, 1 mM EDTA, pH 7.5) was pipetted into a 96-wellplate containing 30 μL purified cellular RNA. The plate was read in aCytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm n andemission at 530 nm.

Probes and primers for use in real-time PCR were designed to hybridizeto target-specific sequences. The primers and probes and LMNA targetnucleic acid sequences to which they hybridize are presented in Table 2.The target-specific PCR probes have FAM covalently linked to the 5′ endand TAMRA or MGB covalently linked to the 3′ end, where FAM is thefluorescent dye and TAMRA or MGB is the quencher dye.

TABLE 2 LMNA-specific primers and probes for use in real-time PCR TargetTarget SEQ Sequence SEQ ID Name Species ID NO Description Sequence (5′to 3′) NO LMNA Human 4 Forward Primer CCTTAAAACCAAAGAGGGCTTC 8 LMNAHuman 4 Reverse Primer CATGTCACAGGGTCCCCG 9 LMNA Human 4 ProbeCTTTTCTGCCCTGGCTGCTGCC 10

EXAMPLE 3 Antisense Inhibition of Human LMNA by Oligomeric Compounds

A series of oligomeric compounds was designed to target differentregions of human LMNA, using published sequences cited in Table 1. Thecompounds are shown in Table 3. All compounds in Table 3 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of 10 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′) by five-nucleotide “wings”. The wingsare composed of 2′-O-(2-methoxyethyl) nucleotides, also known as 2′-MOEnucleotides. The internucleoside (backbone) linkages arephosphorothioate throughout the oligonucleotide. All cytidine residuesare 5-methylcytidines. The compounds were screened for their modulatoryeffect on LMNA mRNA levels by quantitative real-time PCR as described inother examples herein. Data are averages from experiments in which A549cells were treated with the antisense compounds at a concentration of 75nM. Data are expressed as percent inhibition relative to control-treatedcells.

TABLE 3 Inhibition of human mRNA levels by chimericoligonucleotides having 2′-MOE wings and deoxy gap Target SEQ ID Target% SEQ ISIS # NO Site Sequence 5′ to 3′) Inhibition ID NO 366687 1 2001GGGCTCTGGGCTCCTGAGCC 3 11 366773 4 1913 CCGAGCTGCTGCAGTGGGAG 42 12366774 4 1966 GCAGGTCCCGCACAGCACGG 61 13 366775 4 1994TGGCAGATGCCTTGTCGGCA 21 14 366776 4 2001 GAGCCGCTGGCAGATGCCTT 24 15366777 4 2003 CTGAGCCGCTGGCAGATGCC 60 16 366778 4 2030AGGAGATGGGTCCGCCCACC 44 17 366779 4 2033 CAGAGGAGATGGGTCCGCCC 38 18366780 4 2035 GCCAGAGGAGATGGGTCCGC 54 19 366781 4 2037GAGCCAGAGGAGATGGGTCC 43 20 359381 4 2048 TGGAGGCAGAAGAGCCAGAG 50 21366782 4 2052 ACACTGGAGGCAGAAGAGCC 43 22 366783 4 2054TGACACTGGAGGCAGAAGAG 44 23 366784 4 2057 CCGTGACACTGGAGGCAGAA 47 24366785 4 2062 AGTGACCGTGACACTGGAGG 69 25 366786 4 2067CTGCGAGTGACCGTGACACT 67 26 366787 4 2072 GGTAGCTGCGAGTGACCGTG 71 27366788 4 2162 TCTGGGTTCGGGGGCTGGAG 54 28 366789 4 2165GGCTCTGGGTTGGGGGGCTG 3 29 366686 4 2166 GGGCTCTGGGTTCGGGGGCT 13 30366685 4 2171 TCTGGGGGCTCTGGGTTCGG 15 31 366790 4 2174AGTTCTGGGGGCTCTGGGTT 0 32 366791 4 2178 CTGCAGTTCTGGGGGCTCTG 38 33366792 4 2193 CCAGATTACATGATGCTGCA 75 34 366793 4 2202TGGCAGGTCCCAGATTACAT 73 35 366794 4 2462 TTTCGTGGAAGCAGGGAAAA 41 36366795 4 2491 CCCTCTTTGGTTTTAAGGCA 81 37 366796 4 2502TCTAGAGGAAGCCCTCTTTG 45 38 366797 4 2507 TGGCTTCTAGAGGAAGCCCT 15 39366798 4 2509 CTTGGCTTCTAGAGGAAGCC 10 40 366799 4 2529CTATAAAAGCACCCCTTTCC 0 41 366800 4 2538 AGCTAGCCTCTATAAAAGCA 34 42366801 4 2547 AAAAGCAGAAGCTAGCCTCT 67 43 366802 4 2554AGGGCAGAAAAGCAGAAGCT 17 44 366803 4 2595 AGGCACCATGTCACAGGGTC 85 45366804 4 2606 GCCTGCCTCTCAGGCACCAT 51 46 366805 4 2627GGCTGGCGGAGAAGCCTCTA 49 47 366806 4 2647 TGAGCCTGCCGTCCAGAGGA 40 48366807 4 2727 ATCACAGCAGGCCAAGCCCA 33 49 366808 4 2741CCAGGTGTAGTGGAATCACA 73 50 366809 4 2821 TGGTACCTGGGAGAATGGAC 43 51366810 4 2822 CTGGTACCTGGGAGAATGGA 38 52 366811 4 2824AGCTGGTACCTGGGAGAATG 36 53 366812 4 2835 AAAGCAAGCGCAGCTGGTAC 81 54366813 4 2840 ACAGAAAAGCAAGCGCAGCT 84 55 366814 4 2857TCTTGTCTAAATAAAATACA 12 56 366815 4 2860 ATCTCTTGTCTAAATAAAAT 0 57366816 4 2912 GCAGCTCAAACTCACCTTTC 80 58 366817 4 2917GGAAGGCAGCTCAAACTCAC 69 59 366818 4 2921 CTAGGGAAGGCAGCTCAAAC 69 60366S19 4 2943 AGAGCCCACCCAGGGTCTAA 38 61 366820 4 2955CAGTGACTGCACAGAGCCCA 70 62 366821 4 3162 AGAAACAACTAGTGTATTGA 68 63366822 6 2012 TCTGGGCTCCTGAGCCGCTG 26 64 366823 6 2015GGCTCTGGGCTCCTGAGCCG 16 65 366824 6 2020 CTGGGGGCTCTGGGCTCCTG 22 66366825 6 2022 TTCTGGGGGCTCTGGGCTCC 25 67 366826 6 2023GTTCTGGGGGCTCTGGGCTC 27 68 366827 6 2027 TGCAGTTCTGGGGGCTCTGG 43 69366828 6 2031 ATGCTGCAGTTCTGGGGGCT 17 70 366829 7 23651ATGGCATGAGAAAGTTCCCA 59 71 366830 7 23662 AGGAATATTCCATGGCATCA 50 72366831 7 23668 TCCCACAGGAATATTCCATG 63 73 366832 7 23772GATATGCATGCAACAAGAAT 0 74 366833 7 23875 GGAGCAAGCTTGTAGGGCAG 16 75366834 7 23907 CTCTGAGCTTAAGAGGAAAA 22 76 366835 7 24067AGGCAGGAAGGTGTGGTTCT 42 77 366836 7 24158 GAAGGGAGACAAGGCTCAGG 38 78366837 7 24461 GATTTGGAGACAAAGCAGAG 43 79 366838 7 24463AGGATTTGGAGACAAAGCAG 41 80 366839 7 24465 GCAGGATTTGGAGACAAAGC 50 81366840 7 24466 TGCAGGATTTGGAGACAAAG 50 82 366841 7 24472CCCGCCTGCAGGATTTGGAG 47 83 366842 7 24480 ACCAGGGACCCGCCTGCAGG 40 84366843 7 24489 CCCTCGATGACCAGGGACCC 48 85 366844 7 24491ACCCCTCGATGACCAGGGAC 43 86 366845 7 24496 GTCCTACCCCTCGATGACCA 38 87366846 7 24539 CTGCGGAAGAGAAGGCAGGC 31 88

EXAMPLE 4 Antisense Inhibition of Human LMNA by Oligomeric Compounds

A second series of oligomeric compounds was designed to target differentregions of human LMNA, using published sequences cited in Table 1. Thecompounds are shown in Table 4. All compounds in Table 4 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of 10 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′) by five-nucleotide “wings”. The wingsare composed of 2′-O-(2-methoxyethyl) nucleotides, also known as 2′-MOEnucleotides. The internucleoside (backbone) linkages arephosphorothioate throughout the oligonucleotide. All cytidine residuesare 5-methylcytidines. The compounds were screened for their modulatoryeffect on LMNA mRNA levels by quantitative real-time PCR as described inother examples herein. Data are averages from experiments in which T24cells were treated with the antisense compounds at a concentration of100 nM. Data are expressed as percent inhibition relative tocontrol-treated cells.

TABLE 4 Inhibition of human LMNA mRNA levels by chimericoligonucleotides having 2′-MOE wings and deoxy gap Target SEQ ID Target% SEQ ISIS # NO Site Sequence 5′ to 3′) Inhibition ID NO 174126 4 803GTTCCTCCTTCATGGTCTGC 93 89 174127 4 734 TCTTGGCCTCACCTAGGGCT 82 90174128 4 1671 TGCCCAGCCTTCAGGGTGAA 61 91 174129 4 1453GAAGCTGCTGCGGCTGTCAG 75 92 174130 4 1844 CAACCACAGTCACTGAGCGC 76 93174131 4 370 CAGCCCTGCGTTCTCCGTTT 81 94 174132 4 1410GAGTGAGAGGAAGCACGGCC 51 95 174133 4 828 CTGTAGATGTTCTTCTGGAA 63 96174134 4 365 CTGCGTTCTCCGTTTCCAGC 85 97 174135 4 1206AGCAGCCGCCGGGTGGTGTC 56 98 174136 4 1649 TTGGTGGGAACCGGTAAGTC 60 99174137 4 1037 TGTTCCTCTCAGCAGACTGC 86 100 174138 4 1637GGTAAGTCAGCAAGGGATCA 75 101 174139 4 1648 TGGTGGGAACCGGTAAGTCA 59 102174140 4 844 CTCACGCAGCTCGTCACTGT 73 103 174141 4 1759GCAGCCCCAGGTGTTCTGTG 60 104 174142 4 121 GAGACTGCTCGGAGTCGGAG 55 105174143 4 791 TGGTCTGCAGCCTGTTCTCA 93 106 174144 4 1475TGCGGCTCTCAGTGGACTCC 85 107 174145 4 1040 TGCTGTTCCTCTCAGCAGAC 61 108174146 4 851 GCTTGGTCTCACGCAGCTCC 85 109 174147 4 1884TGATGGAGCAGGTCATCTCC 29 110 174148 4 1782 AGAGCCGTACGCAGGCTGTT 49 111174149 4 376 AAGGCGCAGCCCTGCGTTCT 55 112 174150 4 1170GAGTCCTCCAGGTCTCGAAG 60 113 174151 4 572 GGTCACCCTCCTTCTTGGTA 75 114174152 4 257 ACAGCGGAGTGGAGCTGGCC 68 115 174153 4 1597CTGCCAATTGCCCATGGACT 6 116 174154 4 1129 CTGCTTCTGGAGGTGGCTGA 17 117174155 4 549 CGCGCTTTCAGCTCCTTAAA 86 118 174156 4 748ATCCTGAAGTTGCTTCTTGG 75 119 174157 4 465 GTCTTGCGGGCATCCCCGAG 64 120174158 4 385 GGTGATGCGAAGGCGCAGCC 71 121 174159 4 1491TGCTGTGAGAAGCTGCTGCG 77 122 174160 4 1428 CCACCCTGTGTCTGGGATGA 82 123174161 4 1645 TGGGAACCGGTAAGTCAGCA 64 124 174162 4 442GGCCTCGTAGGCGGCCTTGA 54 125

EXAMPLE 5 Dose-Dependent Inhibition of Human LMNA by OligomericCompounds

To further evaluate LMNA antisense compounds, ISIS 174134, ISIS 174137,ISIS 174146, ISIS 366812, ISIS 366813 and ISIS 366816 and a controloligonucleotide were tested in A549 cells at doses of 0.4, 1.2, 3.7, 11,33 and 100 nM. The compounds were analyzed for their effect on LMNA mRNAlevels by quantitative real-time PCR as described in other examplesherein.

Table 5 shows the reduction in expression at each dose, expressed aspercent inhibition relative to untreated control. If the targetexpression level of oligomeric compound-treated cell was higher thancontrol, percent inhibition is expressed as zero inhibition.

TABLE 5 Dose-dependent inhibition of LMNA ISIS # 0.4 nM 1.2 nM 3.7 nM 11nM 33 nM 100 nM 174134 0 16 32 43 59 62 174137 0 0 0 0 42 48 174146 0 525 38 62 65 366812 0 0 0 17 49 75 366813 6 0 17 32 37 71 366816 0 0 4 3858 70 Control 4 0 8 14 0 0

Each of the LMNA antisense compounds demonstrated a dose-dependentinhibition of LMNA mRNA expression.

EXAMPLE 6 Modified LMNA Oligomeric Compounds for Modulation of Splicing

Hutchinson-Gilford progeria syndrome is caused by spontaneous pointmutations in LMNA, the most commonly reported of which is a GGC to GGTchange at codon 608. This mutation resides in exon 11 of LMNA andresults in activation of a cryptic splice site four nucleotidesupstream. When the aberrant splice donor site is spliced to the normalexon 12 acceptor site, a truncated LMNA mRNA is produced which lacks 150nucleotides of exon 11. The truncated mRNA produces a truncated proteinlacking 50 amino acids in the globular tail domain. The truncatedprotein accumulates as farnesyl-prelamin A and cannot be processed tothe mature form of the protein, which results in toxicity to the celland HGPS disease phenotypes.

To modulate splicing of mutant LMNA pre-mRNA, a series of antisensecompounds was designed to block the aberrant splice site and promote useof the normal exon 11 splice site. The coding sequence for human LMNA isprovided as SEQ ID NO: 4. Exon 11 of normal LMNA includes nucleotides1911 to 2180 of SEQ ID NO: 4, while the truncated form of LMNA found inHGPS patients ends at nucleotide 2130. Each of the compounds, shown inTable 6, is uniformly modified with 2′-O-(2-methoxyethyl) nucleotides ateach position. The internucleoside (backbone) linkages arephosphorothioate throughout the oligonucleotide. All cytidine residuesare 5-methylcytidines. The compounds vary in length from 16 to 20nucleotides. ISIS 355076 and ISIS 355077 were designed to contain asingle mismatch relative to the target sequence (SEQ ID NO: 4) in orderto be 100% complementary to LMNA sequences containing one of twomutations associated with HGPS (GGC to GGT or GGC to AGC at codon 608).

TABLE 6 Modified LMNA oligomeric compounds for modulation of splicingTarget SEQ ID Target SEQ ISIS # NO Site Sequence (5′ to3′) ID NO 3550674 2021 GTCGGCCCACCTGGGCTCCT 126 355068 4 2012 CCTGGGCTCCTGAGCCGCTG 127355069 4 2001 GAGCCGCTGGCAGATGCCTT 128 355070 4 1991CAGATGCCTTGTCGGCAGGC 129 355071 4 2024 TGGGTCCGCCCACCTGGGCT 130 355072 42035 CCAGAGGAGATGGGTCCGC 131 355073 4 2048 TGGAGGCAGAAGAGCCAGAG 132355074 4 2064 AGTGACCGTGACACTGGA 133 355075 4 2026 GGTCCGCCCACCTGGG 134355076 4 2026 GGTCCACCCACCTGGG 135 355077 4 2026 GGTCCGCTCACCTGGG 136

The compounds were analyzed for their effect on LMNA mRNA levels infibroblast cell lines obtained from four different HGPS patients(Coriell Cell Repository, Camden, N.J.). Two of the cell lines (AG01972and AG03513E) are derived from patients known to contain the GGC to GGTchange at codon 608. The other two cell lines (AG11513A and AG06917B)are derived from HGPS patients with an unknown mutation. A fibroblastcell line derived from a normal parent (AG06299B) was used as a controlcell line. Fibroblasts were treated with 200 nM of oligonucleotide for24 hours. LMNA splice products were analyzed by RT-PCR using primersflanking exon 11 and subjected to electrophoresis using methods wellknown in the art.

In HGPS patient cell lines treated with uniformly modified antisensecompounds, several different effects were observed. In at least onepatient cell line, ISIS 355069 and ISIS 355070 appeared to increase theratio of normal LMNA mRNA to truncated mRNA. However, treatment withISIS 355074 resulted in a significant increase in truncated LMNA mRNA inboth HGPS cell lines and in the normal cell line. This result suggeststhat ISIS 355074 may target a splice enhancer element required forefficient splicing of the natural LMNA product. Thus, ISIS 355074effectively inhibited the use of the natural splice site resulting in anincrease in use of the cryptic splice site, even in normal cells.

EXAMPLE 7 Identification of Splicing Enhancer Elements and SplicingSilencer Elements Using Modified Antisense Compounds Modulation of LMNASplicing Products

Cis-acting regulatory enhancer and silencer elements, found in exons andintrons, play an important role in directing splicing of eukaryoticmRNAs. Exonic splicing enhancer elements, for example, are known toserve as binding sites for SR proteins, which promote splicing byrecruiting spliceosomal components. Consensus binding sequences forseveral SR proteins have been determined (Cartegni et al 2003, NucleicAcids Res. 31(13):3568-3571). Less is known about trans factors thatbind intronic splicing elements, but studies have suggested SR proteinsand heterogeneous nuclear ribonucleoproteins (hnRNPs) may play a role inregulating splicing through interaction with intronic elements (Yeo etal. 2004, Proc. Natl. Acad. Sci. U.S.A. 101(44):15700-15705).Identification of both intronic and exonic splicing elements is usefulfor developing therapeutic tools to modulate the splicing process.

Sequence analysis of the binding site for ISIS 355074 reveals aconsensus binding sequence (TGTCACG) for the SR protein SRp40 (Cartegniet al. 2003, Nucleic Acids Res. 31(13):3568-3571) at nucleotides2069-2075 of SEQ ID NO: 4. A second SRp40 consensus site (TCACTCG) isalso present one nucleotide downstream (3′) of the first (nucleotides2077-2083 of SEQ ID NO: 4), a portion of which is contained within theISIS 355074 binding site. The SRp40 binding sites are upstream of thenormal LMNA splice donor site, indicating they play a role in promotinguse of the normal splice site, resulting in production of normal LMNAmRNA. This finding, in combination with the results shown in Example 6,suggest that blocking a SRp40 site(s) with modified antisense compoundssuch as ISIS 355074 can block binding of SRp40, thus promoting use ofthe cryptic splice site and generating truncated LMNA mRNA. To furtherevaluate the effect of modified compounds targeting this region of LMNA,a set of antisense compounds was designed as a micro-walk around theISIS 335074 binding site. The micro-walk compounds target an exonicregion up to about 150 nucleotides upstream (5′) of the normal LMNAsplice donor site. The sequence and target site (the 5′-most nucleotideof the target nucleic acid to which the compound binds) of themicro-walk compounds are shown in Table 7. Each of the compounds is 18nucleobases in length and is uniformly modified with2′-O-(2-methoxyethyl) nucleotides at each position. The internucleoside(backbone) linkages are phosphorothioate throughout the oligonucleotide.All cytidine residues are 5-methylcytidines. Bold residues indicatethose that overlap with ISIS 355074.

TABLE 7 Modified compounds designed for ISIS 355074 micro-walk TargetSEQ ID Target SEQ ISIS # NO Site Sequence (5′ to 3′) ID NO 385317 4 2053CACTGGAGGCAGAAGAGC 137 385318 4 2055 GACACTGGAGGCAGAAGA 138 385319 42057 GTGACACTGGAGGCAGAA 139 385320 4 2059 CCGTGACACTGGAGGCAG 140 3853214 2061 GACCGTGACACTGGAGGC 141 355074 4 2064 AGTGACCGTGACACTGGA 133385322 4 2065 GAGTGACCGTGACACTGG 142 385323 4 2067 GCGAGTGACCGTGACACT143 385324 4 2069 CTGCGAGTGACCGTGACA 144 385325 4 2071AGCTGCGAGTGACCGTGA 145 385326 4 2073 GTAGGTGCGAGTGACCGT 146 385327 42075 CGGTAGCTGCGAGTGACC 147 385328 4 2077 TGCGGTAGCTGCGAGTGA 148 3853294 2079 ACTGCGGTAGCTGCGAGT 149 385330 4 2081 AGACTGCGGTAGCTGCGA 150385331 4 2083 CCACACTGCGGTAGCTGC 151

Shown in Table 8 is the target sequence (SEQ ID NO: 4) for eachmicro-walk compound. Residues comprising the first SRp40 consensussequence (TGTCACG) are shown in bold and residues comprising the secondSRp40 consensus sequence (TCACTCG) are underlined.

TABLE 8 Target sequences of ISIS 355074 micro-walk compounds TargetTarget Sequence nucleotides SEQ ISIS # (5′ to 3′) (SEQ ID NO:4) ID NO385317 GCTCTTCTGCCTCCAGTG 2053-2070 162 385318 TCTTCTGCCTCCAGTGTC2055-2072 163 385319 TTCTGCCTCCAGTGTCAC 2057-2074 164 385320CTGCCTCGAGTGTCACGG 2059-2076 165 385321 GCCTCCAGTGTCACGGTC 2061-2078 166355074 TCCAGTGTCACGGTCACT 2064-2081 167 385322 CCAGTGTCACGGTCACTC2065-2082 168 385323 AGTGTCACGGTCACTCGC 2067-2084 169 385324TGTCACGGTCACTCGCAG 2069-2086 170 385325 TCACGGTCACTCGCAGCT 2071-2088 171385326 ACGGTCACTCGCAGCTAC 2073-2090 172 385327 GGTCACTCGCAGGTACCG2075-2092 173 385328 TCACTCGCAGCTACCGCA 2077-2094 174 385329ACTCGCAGCTACCGCAGT 2079-2096 175 385330 TCGCAGCTACCGCAGTGT 2081-2098 176385331 GCAGCTACCGCAGTGTGG 2083-3000 177

The compounds were evaluated for their effect on LMNA splicing in normalfibroblasts. Cells were transfected with 200 nM of each compound shownin Table 7 and RNA was isolated after 24 h. RT-PCR was performed usingprimers flanking LMNA exon 11 and the products were subjected toelectrophoresis using methods well known in the art. In untreated cellsor cells treated with a control oligonucleotide, only the normal LMNAmRNA product was detected, as expected. Treatment with the remainder ofthe LMNA compounds resulted in a mixture of the normal (long form) andtruncated (short form) of LMNA mRNA. ISIS 385321, ISIS 385322, ISIS385323, ISIS 385324, ISIS 385325, ISIS 385326, ISIS 385327, ISIS 385328and ISIS 355074 promoted production of the truncated form LMNA mosteffectively. In particular, little to no normal LMNA mRNA was detectedfollowing treatment with ISIS 355074, ISIS 385324 or ISIS 385325. Thecompounds most effective at promoting the short form of LMNA mRNAappeared to be those that completely bound one SRp40 consensus site andat least a portion of the other SRp40 consensus site. ISIS 385324 iscompletely complementary to both of the consensus sites, ISIS 385325binds the second consensus site and 5 of 7 nucleotides of the firstconsensus site while ISIS 355074 binds the first consensus site and 5 of7 nucleotides of the second site. These results demonstrate thatantisense compounds that target a splicing enhancer element for thenormal LMNA splice site are capable of modulating the ratio of LMNAsplice products to favor the truncated form of LMNA by promoting usageof the cryptic splice site.

To determine whether the LMNA compounds targeting SRp40 consensus sitesactually inhibit binding of SRp40 to LMNA mRNA, an ELISA was performedusing standard procedures. Normal fibroblasts were either untreated ortreated with 10 μM ISIS 385331, ISIS 385324, ISIS 385325 or ISIS 355074for 48 h. Cell extracts were prepared and incubated with an SRp40consensus sequence. Binding of SRp40 to LMNA mRNA in the cell extractswas evaluated by ELISA. The results are shown in Table 9 as percentSRp40 binding relative to untreated control.

TABLE 9 Detection of SRp40 binding in cell extracts treated with LMNAcompounds % SRp40 Treatment Binding Untreated 100 ISIS 385331 80 ISIS385324 67 ISIS 385325 46 ISIS 355074 77

The results of the SRp40 binding assay are in accordance with the levelsof the long and short forms LMNA mRNA observed, suggesting that themodified LMNA antisense compounds alter splicing by inhibiting bindingof SRp40.

Next, to determine whether modified compounds could also be designed topromote production of normal LMNA mRNA by targeting (and thus blocking)splicing enhancers for the cryptic splice donor site, a series ofcompounds was designed to target the region upstream of the crypticsplice site (nucleotides 2030-2036 of SEQ ID NO: 4), which is activatedin progeria patients. The compounds target an exonic region up to about150 nucleotides upstream (5′) of the cryptic splice donor site. Each ofthe compounds, shown in Table 10, is 20 nucleobases in length and isuniformly modified with 2′-O-(2-methoxyethyl) nucleotides at eachposition. The internucleoside (backbone) linkages are phosphorothioatethroughout the oligonucleotide. All cytidine residues are5-methylcytidines.

TABLE 10 Modified LMNA compounds targeting theregion upstream of the LMNA mRNA exon 11 cryptic splice acceptor siteTarget SEQ ID Target SEQ ISIS # NO Site Sequence (5′ to 3′) ID NO 3863584 1906 GCTGCAGTGGGAGCCGTGGT 152 386359 4 1908 CTGCTGCAGTGGGAGCCGTG 153386360 4 1911 GAGCTGCTGCAGTGGGAGCC 154 3S6361 4 1916CCCCCGAGCTGCTGCAGTGG 155 386362 4 1920 GGGTCCCCCGAGCTGCTGCA 156 3S6363 41931 TGTACTCAGCGGGGTCCCCC 157 386364 4 1938 CGCAGGTTGTACTCAGCGGG 158386365 4 1971 TGCCCGCAGGTCCCGCACAG 159 386366 4 1976CAGGCTGCCCGCAGGTCCCG 160 386367 4 1981 GTCGGCAGGCTGCCCGCAGG 161

The compounds were evaluated for their effect on LMNA splicing inprogeria fibroblasts. Cells were transfected with 200 nM of eachcompound shown in Table 10. Untreated cells and cells treated with acontrol oligonucleotide or ISIS 355074 were included as controls. RNAwas isolated after 24 h. RT-PCR was performed using primers flankingLMNA exon 11 and the products were subjected to electrophoresis usingmethods well known in the art. In untreated cells and cells treated witha control oligonucleotide, a mixture of the long and short form of LMNAmRNA was observed, as expected for progeria fibroblasts. Supporting theresults shown above, treatment with ISIS 355074 resulted in productionof only the short form of LMNA mRNA. Treatment with ISIS 386357, ISIS386358, ISIS 386359, ISIS 386360, ISIS 386361, ISIS 386362, ISIS 386363and ISIS 386364 resulted in little to minor changes in the ratio of longform to short form of LMNA mRNA and fibroblasts treated with ISIS 386367appeared to show a modest increase in levels of the long form of LMNAmRNA. In contrast, treatment with ISIS 386365 or ISIS 386366 resulted ina significant shift toward production of the long form of LMNA mRNA.These results demonstrate that ISIS 386365 and ISIS 386366 modulated theratio of splicing products to favor the normal form by promoting usageof the normal splice donor site.

Sequence analysis of the region upstream of the LMNA cryptic splice sitereveals an SC35 consensus binding site (GACCTGCG) at nucleotides1979-1986 of SEQ ID NO: 4. Shown in Table 11 is the target sequence (SEQID NO: 4) for each compound listed in Table 10. Residues comprising theSC35 consensus sequence are shown in bold.

TABLE 11 Target sequences of compounds targeting theregion upstream of the cryptic splice site Target SEQ Target Sequencenucleotides ID  ISIS # (5′ to 3′) (SEQ ID NO:4) NO 386358ACCAGGGCTCCCACTGCAGC 1906-1925 178 3S6359 CACGGCTCCCACTGCAGCAG 1908-1927179 386360 GGCTCCCACTGCAGCAGCTC 1911-1930 180 386361CCACTGCAGCAGCTCGGGGG 1916-1935 181 386362 TGCAGCAGCTCGGGGGACCC 1920-1939182 386363 GGGGGACCCCGCTGAGTACA 1931-1950 183 386364CCCGCTGAGTACAACCTGCG 1938-1957 184 386365 CTGTGCGGGACCTGCGGGCA 1971-1990185 386366 CGGGACCTGCGGGCAGCCTG 1976-1995 186 386367CCTGCGGGCAGCCTGCCGAC 1981-2000 187

Both ISIS 386365 and ISIS 386366, which were the most effectivecompounds at altering splicing toward production of the long form ofLMNA mRNA, target a region that encompasses the SC35 consensus sequence.Furthermore, ISIS 386367, which also increased the level of normal LMNAmRNA, is complementary to 6 of the 8 nucleotides of the SC35 consensussequence. These findings suggest that modified compounds targeting theSC35 consensus sequence inhibit use of the cryptic splice site, drivingsplicing toward production of the long form of LMNA mRNA by promotingusage of the normal splice site. These results also indicate thatmodified oligonucleotides can be effectively and efficiently used toidentify splicing elements such as splicing enhancer elements andsplicing silencer elements.

Previous studies have shown that tissue inhibitor of metalloproteinase 3(TIMP3) is downregulated in fibroblasts of patients diagnosed withprogeria (Csoka et al. 2004, Aging Cell 3:235-243) and that correctionof LMNA aberrant splicing in these cells restores normal expressionlevels of TIMP3 (Scaffidi and Misteli, 2005, Nature Med. 11(4):440-445).To further evaluate the phenotypic effect of the modified LMNAcompounds, progeria fibroblasts were treated with selected compounds andexpression levels of TIMP3 were determined by RT-PCR using standardprocedures. Progeria fibroblasts were either untreated or treated with180 nM of ISIS 386365, ISIS 386366 or ISIS 386358 for 72 h. Untreatednormal fibroblasts were used as a control. Expression levels of TIMP3are shown in Table 12 as percent mRNA expression relative to untreatedprogeria fibroblasts.

TABLE 12 Effect of LMNA antisense compounds on TIMP3 expression inprogeria fibroblasts % TIMP3 Fibroblasts Treatment expression NormalUntreated 343 Progeria Untreated 100 Progeria ISIS 386365 204 ProgeriaISIS 386366 340 Progeria ISIS 386358 141

As previously reported, the level of TIMP3 expression was significantlyreduced in untreated progeria fibroblasts relative to untreated normalfibroblasts. ISIS 386258, which had little to no effect on modulatingthe ratio of LMNA splicing products, led to a slight increase in thelevel of TIMP3 expression in progeria fibroblasts. However, treatmentwith ISIS 386365 and ISIS 386366, which significantly promoted splicingof the long form of LMNA mRNA, significantly increased the level ofTIMP3 expression in progeria fibroblasts, with ISIS 386366 essentiallyrestoring TIMP3 expression to a normal level. These results demonstratethat modified compounds targeting a splicing enhancer element for thecryptic splice site found in progeria patients can effectively promotesplicing of the normal LMNA mRNA and restore normal cellular phenotype.

1. A method of modulating splicing of a LMNA pre-mRNA in a cell,comprising contacting the cell with an oligomeric compound comprising anoligonucleotide consisting of 12 to 30 linked nucleosides, wherein theoligonucleotide has a nucleobase sequence comprising an at least 8nucleobase portion complementary to an equal length portion ofnucleotides 1971-2050, 2015-2094 or 2013-2092 of SEQ ID NO: 4 and is atleast 90% complementary to LMNA pre-mRNA having SEQ ID NO:4 as measuredover the entirety of the oligonucleotide, wherein each nucleoside of theoligonucleotide comprises a modified sugar moiety; and therebymodulating splicing of the LMNA pre-mRNA in the cell.
 2. The method ofclaim 1, wherein the modulation of splicing results in an increase inthe ratio of full-length LMNA mRNA to truncated LMNA mRNA.
 3. The methodof claim 1, wherein the modulation of splicing results in an increase inthe ratio of full-length lamin A protein to truncated lamin A protein.4. The method of claim 1, wherein each nucleoside of saidoligonucleotide comprises the same modified sugar moiety.
 5. The methodof claim 1, wherein each nucleoside of said oligonucleotide comprises a2′-O-(methoxyethyl) modified sugar moiety.
 6. The method of claim 1,wherein the cell is in an animal.
 7. The method of claim 6, wherein theanimal is a human.
 8. The method of claim 1, wherein at least oneinternucleoside linkage of said oligonucleotide is a modifiedinternucleoside linkage.
 9. The method of claim 1, wherein eachinternucleoside linkage of said oligonucleotide is a phosphorothioateinternucleoside linkage.
 10. The method of claim 1, wherein at least onenucleoside of said oligonucleotide comprises a modified nucleobase. 11.The method of claim 10, wherein the modified nucleobase is a5-methylcytosine.
 12. The method of claim 1, wherein each cytosine insaid oligonucleotide is a 5-methylcytosine.
 13. The method of claim 1,wherein each nucleoside of the oligonucleotide comprises a2′-O-(methoxyethyl) modified sugar moiety, wherein each internucleosidelinkage of said oligonucleotide is a phosphorothioate internucleosidelinkage, and wherein each cytosine in said oligonucleotide is a5-methylcytosine.
 14. The method of claim 1, wherein saidoligonucleotide consists of 15 to 30 linked nucleosides.
 15. The methodof claim 1, wherein the oligonucleotide has a nucleobase sequencecomprising an at least 8 nucleobase portion complementary to an equallength portion of nucleotides 1971-2050 of SEQ ID NO:
 4. 16. The methodof claim 1, wherein the oligonucleotide has a nucleobase sequencecomprising an at least 8 nucleobase portion complementary to an equallength portion of nucleotides 2013-2092 of SEQ ID NO:
 4. 17. The methodof claim 1, wherein the oligonucleotide has a nucleobase sequencecomprising an at least 8 nucleobase portion complementary to an equallength portion of nucleotides 2015-2094 of SEQ ID NO: 4.